The Legionella collagen-like protein employs a distinct binding mechanism for the recognition of host glycosaminoglycans

Bacterial adhesion is a fundamental process which enables colonisation of niche environments and is key for infection. However, in Legionella pneumophila, the causative agent of Legionnaires’ disease, these processes are not well understood. The Legionella collagen-like protein (Lcl) is an extracellular peripheral membrane protein that recognises sulphated glycosaminoglycans on the surface of eukaryotic cells, but also stimulates bacterial aggregation in response to divalent cations. Here we report the crystal structure of the Lcl C-terminal domain (Lcl-CTD) and present a model for intact Lcl. Our data reveal that Lcl-CTD forms an unusual trimer arrangement with a positively charged external surface and negatively charged solvent exposed internal cavity. Through molecular dynamics simulations, we show how the glycosaminoglycan chondroitin-4-sulphate associates with the Lcl-CTD surface via distinct binding modes. Our findings show that Lcl homologs are present across both the Pseudomonadota and Fibrobacterota-Chlorobiota-Bacteroidota phyla and suggest that Lcl may represent a versatile carbohydrate-binding mechanism.

Supplementary Figure 4.| Solution NMR spectroscopy analyses of 13 C 15 N glycine labelled CLR peptide. 1 H-1 H NOESY spectra (mixing time 240 ms) recorded at 275 K (black) and 310 K (blue), and 1 H-1 H ROESY (mixing time 200 ms) spectrum recorded at 275 K (red) expanded on the amide region.Significant differences are observed between the NOESY and ROESY spectra at 275 K and significant spectral broadening is observed in the NOESY spectrum at 310 K.This indicates that the peptide is in equilibrium between monomeric and trimeric states.

SASD-UQ7
Supplementary  b The frequency was calculated as average over all the replicas (production only).When more than one donor/acceptor per residue was involved in hydrogen bonding, the frequency values were added together.Only residues with a frequency >= 10% for at least one chain are reported.

Supplementary Figure 5 .Supplementary Figure 6 .
Circular dichroism (CD) spectra of Lcl. a CD spectra of Lcl recorded at 10°C with negative peak at 199 nm and maximum peak at 222 nm.b Thermal denaturation of Lcl recorded between 10°C and 75°C at 199 nm.Two melting temperatures were determined at 38°C and 45°C.Source data are provided as an accompanying Source Data file.Structures of Lcl-CTD and Lcl-CTD/SO4.a Topology diagram showing an individual Lcl-CTD domain.-helix and 310-helix secondary structure are coloured orange, β-sheet secondary structure is coloured green, and loops are grey.Helices are labelled 1-2 and β-sheets are labelled 1-9.b,c An example of the electron-density quality of the Lcl-CTD and Lcl-CTD/SO4 structures.Residues Asn362 to Lys369 of chain A are shown as sticks with a σA-weighted 2Fo -Fc map contoured at 1.5 rms electron density.d Cross-eye stereo view of an omit map contoured at 1.5 rms electron density, for the two bound SO4 2-ions in the Lcl-CTD/SO4 structure.Direct and indirect hydrogen bonding (red dashed line) is observed between the SO4 2-ions and Lys369, Ser370, Asn373, Asp386 and Ala388 in chain A (sticks).Supplementary Figure 7. SAXS analysis of Lcl-CTD.a Experimental scattering curve of Lcl-CTD (black open circles).Inset: Guinier region (orange open circles) and linear regression (black line) for Rg evaluation.b Shape distribution [P(r)] function derived from SAXS analysis for Lcl-CTD.c Kratky plot indicates that Lcl-CTD has dynamic properties in solution.Supplementary Figure 8. SAXS derived model of Lcl-CTD in solution.a EOM yielded three distinct populations: extended N-terminus (50%), partially extended N-terminus (20%) and compact Nterminus (30%).Lcl-CTD models corresponding to the centre of each population are shown.b EOM (red line) and crystal structure (blue) fit to the Lcl-CTD SAXS data (black open circles) with χ 2 of 1.05 and 93.75, respectively.Source data are provided as an accompanying Source Data file.Supplementary Figure 9. Structural comparison of the Lcl-CTD monomer with other C-type lectin-like domains.Homologous structures identified using the Dali server with Z-scores >8.0 are shown 2 .a Cartoon representation of Lcl-CTD (green) superimposed on the platelet glycoprotein Ib-α binding protein mucrocetin from the venom of Trimeresurus mucrosquamatus (teal; Protein Data Bank (PDB) ID code 1v4l 3 ; chain B residues 201-325; Z-score 9.7; root mean squared deviated (rmsd) 2.7 Å). b Cartoon representation of Lcl-CTD (green) superimposed on the integrin binding protein EMS16 from the venom of Echis multisquamatus (yellow; PDB ID code 1v7p 4 ; chain A residues 1-127; Zscore 9.1; rmsd 2.7 Å). c Cartoon representation of Lcl-CTD (green) superimposed on the integrin binding protein rhodocetin from the venom of Calloselasma rhodostoma (blue; PDB ID code 6nd8; chain B residues 1-122; Z-score 8.5; rmsd 2.8 Å). d Cartoon representation of Lcl-CTD (green) superimposed on the adhesin protein intimin from enteropathogenic Escherichia coli (orange; PDB ID code 1f00 5 ; chain I residues 840-939; Z-score 8.3; rmsd 3.5 Å). e Cartoon representation of Lcl-CTD (green) superimposed on the integrin binding protein invasin from Yersinia pseudotuberculosis (purple; PDB ID code 1cwv 6 ; chain A residues 887-986; Z-score 8.1; rmsd 2.9 Å).Supplementary Figure 12.SAXS analysis of Lcl-CTD mutants.a Size-exclusion chromatography coupled with SAXS profile of wild-type and engineered Lcl-CTD.b Experimental scattering curves of trimeric forms of wild-type and engineered Lcl-CTD.c DAMMIF bead model of monomeric Lcl-CTD R342A superimposed with chain A from Lcl-CTD, with normalized spatial discrepancy (NSD) score of 1.5.d Monomer Lcl-CTD crystal structure fit to monomeric Lcl-CTD R342A SAXS data (red open circles) with χ 2 of 1.06.Experimental scattering curve of trimeric wild-type Lcl-CTD (black open circles) is shown for comparison.e Cartoon of Lcl-CTD trimer highlighting inter-chain hydrogen bonding between Arg342 (chain A) and Asp316 and Asp319 (chain B).

Table 6 . Average frequency of occurrence of Lcl-CTD/C4S contacts during MD simulations.
The frequency was calculated as average over all the replicas (production only).Only residues with a frequency ≥ 10% for at least one chain are reported.
a Residues are sorted by decreasing average frequency.Lysines are highlighted in bold.b

Table 7 . Average frequency of occurrence of Lcl-CTD/C4S hydrogen bonds during MD simulations.
Residues are sorted by decreasing average frequency.Lysines are highlighted in bold.Hydrogen bonding interactions were detected using Visual Molecular Dynamics 8 with a Donor-Acceptor distance threshold of 3.5 Å and a Hydrogen-Donor-Acceptor angle of 30 o . a